Fas (APO-1 or CD95) is a cell surface receptor associated with the regulation of apoptotic cell death in cells of the immune system. The expression and function of Fas protein in developing thymocytes in the human thymus, but is not function in signaling cell death. An understanding of the human species specific role of Fas in developing thymocytes would not only illuminate how these cells escape apoptotic death signaling through this receptor, but might suggest potential immunomodulating therapies for diseases in which apoptotic signaling through Fas is dysregulated. The mechanism(s) which are responsible for the resistance of human thymocytes to Fas-induced apoptosis will be explored in three concurrent and overlapping approaches. First, apoptotic signal steps which are specifically deficient in human thymocytes will be localized by testing for the competence of the distal Fas signal pathway which is shared with other apoptotic stimuli. TNFalpha, TRAIL, and FAS/FLICE chimeras will be employed to trigger apoptosis in human thymocytes under conditions which involve or short circuit FADD. Second, Fas receptors cloned from human thymocytes will be tested for their intrinsic competence to bind Fas ligand and to multimerize FADD, and for their ability to serve as dominant negative inhibitors of wild type Fas. Thirdly, a human thymocyte cDNA library will be screened for proteins with anti- apoptotic function through a "death trap" approach. These experiments will complement studies of other signal function in other projects of this program project to define the signaling factors which instructed the development of immune cells in the human thymus.